This invention relates to nucleic acid and amino acid sequences of B cell receptor associated proteins and to the use of these sequences in the diagnosis, prevention, and treatment of immunological disorders and cancer.
The B-cell response to antigens, which is modulated through receptors, is an essential component of the normal immune system. Immature B cells undergo a selection process based on antigen binding prior to leaving the bone marrow. Mature B cells recognize foreign antigens through B cell receptors (BCR) and produce specific antibodies which bind the foreign antigens. To generate an efficient response to complex antigens, the BCR, BCR associated proteins, and T cell assistance are required. The antigen/receptor complex is internalized, and the antigen is proteolytically processed. A small part of the antigen remains complexed with major histocompatability complex-II (MHCII) molecules on the surface of the B cells where the complex can be recognized by T cells. T cells activated by antigen presentation secrete a variety of lymphokines that induce B cell maturation.
Signaling through the BCR plays an important role in both the generation of antibody and in the establishment of immunological tolerance. Immature B cells that bind self-antigens while still in the bone marrow are eliminated by apoptosis. In contrast, antigen binding on mature B cells results in activation, proliferation, anergy, or apoptosis. The particular functional response depends on whether the B cell receives co-stimulatory signals through other surface receptors and which signal transduction pathways are activated. A mature B cell co-expresses two classes of membrane-bound immunoglobin, IgM and IgD, which have identical cytoplasmic domains and identical antigen binding specificity. In order to be expressed on the B cell surface, the BCR, whether it is of the IgM or IgD class, must be associated with two other polypeptides, Ig-xcex1 and Ig-xcex2. The cytoplasmic portions of the IgM/Ig-Ig-xcex1/Ig-xcex2 and the IgD/Ig-Ig-xcex1/Ig-xcex2 complex should therefore be identical to each other.
Both Ig-xcex1 and Ig-xcex2 contain a sequence, ITAM (SEQ ID NO:9), that can cause activation of protein tyrosine kinases. In addition, the cytoplasmic tails of Ig-xcex1 and Ig-xcex2 are sufficient for the internalization of the surface proteins and their targeting to endosomal compartments for processing. Early biochemical events in signal transduction, such as protein kinase activation and release of calcium ions, are similar for the two receptors (IgM and IgD); however their subsequent biological effects, however, are different. Antigen binding or cross-linking of the IgM receptor leads to apoptosis, while binding of IgM and IgD, or IgD alone, does not. Binding to IgD alone induces cell proliferation. Analysis of IgD-deficient mice shows that the absence of IgD reduces the efficacy of B cell participation in immune responses. Further in vitro differences in antibody responses, immunological memory, and tolerance have also been described (Carsetti, R. et al. (1993) Eur. J. Immunol. 23: 168-178; Roes, J. et al. (1 993) J. Expt. Med. 177: 45-55; and Kim, K. M. et al. (1992) J. Immunol. 148: 29-34).
Additional analysis of the receptor complex shows that at least five more proteins are associated with the mouse BCR. B cell associated proteins (BAPs) with a mass of 31, 37, and 41 kDa are specifically associated with IgM, while 29 and 31 kDa BAPs are preferentially associated with IgD. BAP 41 has not been characterized, but BAP 32 and BAP 37 are related to prohibitin, a protein which has been implicated in the control of cell proliferation and may have tumor suppressor activity. Both BAP 32 and 37 interact with IgM via its transmembrane domain and contain a C-terminal NPXY (SEQ ID NO:10) motif associated with internalization of proteins. The IgD-associated BAP 29 and BAP 31 show structural features suggesting that they co-localize with the membrane IgM BCR in the plasma membrane. Northern analysis shows that BAP 29, BAP 31, BAP 32, and BAP 37 are expressed in all tissue types examined, and that BAP 32 and BAP 37 are expressed more strongly in transformed cell lines than in normal tissues (Kim, K.-M., et al. (1994) EMBO J.13: 3793-3800; and Terashima, M., et al. (1994) EMBO J.13: 3782-3792).
The discovery of new B-cell receptor associated proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of cancer and immunological disorders.
The present invention features a B-cell receptor-associated protein hereinafter designated BCRP1 and characterized as having similarity to mouse BAP 29.
Accordingly, the invention features a substantially purified BCRP1 having the amino acid sequence shown in SEQ ID NO:1.
One aspect of the invention features isolated and substantially purified polynucleotides that encode BCRP1. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2.
The invention also relates to a polynucleotide sequence comprising the complement of SEQ ID NO:2 or variants thereof. In addition, the invention features polynucleotide sequences which hybridize under stringent conditions to SEQ ID NO:2.
The present invention additionally features a B-cell receptor-associated protein hereinafter designated BCRP2 and characterized as having similarity to mouse BAP 37.
Accordingly, the invention features a substantially purified BCRP2 having the amino acid sequence shown in SEQ ID NO:3. The invention also features a polypeptide variant of BCRP2, SEQ ID NO:5, where F91 and F96 are replaced by S, and V126 is replaced by L.
One aspect of the invention features isolated and substantially purified polynucleotides that encode BCRP2 and the BCRP2 variant (SEQ ID NO:4 and SEQ ID NO:6).
The invention also features polynucleotide sequences comprising the complement of SEQ ID NO:4, SEQ ID NO:6, or variants thereof. In addition, the invention features polynucleotide sequences which hybridize under stringent conditions to SEQ ID NO:4 and SEQ ID NO:6.
The invention additionally features fragments or portions of the nucleic acid sequences encoding the claimed polypeptides, and expression vectors and host cells comprising polynucleotides that encode BCRP. The present invention also features antibodies which bind specifically to BCRP, and pharmaceutical compositions comprising substantially purified BCRP. The invention also features the use of agonists and antagonists of BCRP. The invention also features a method for treating cancer using antagonists of BCRP1, BCRP2, and agonists of BCRP2; for treating immunological disorders using antagonists of BCRP1; and for treating disorders associated with cell growth and differentiation using BCRP 1.